RESUMO
Globular proteins can be decomposed into several modules or secondary structure units. It is useful to investigate the functions of such structural units in order to understand the folding units of proteins. In our previous work, barnase was divided into six peptide fragments corresponding to modules, and some of them were shown to have RNA-binding and RNase activity [Yanagawa, et al. (1993) J. Biol. Chem. 268, 5861-5865]. Barnase mutant proteins obtained by permutation of the structural units also had RNase activity [Tsuji, T. et al. (1999) J. Mol. Biol. 286, 1581-1596]. Here we investigated the structure and function of peptide fragments corresponding to secondary structure units of barnase. The results of circular dichroism spectroscopy indicated that some of the peptide fragments form helical structures in aqueous solutions containing over 30% 2,2,2-trifluoroethanol, and the S6 (94-110) peptide fragment is induced to form a beta-sheet structure in the presence of RNA. The S6 peptide fragment forms aggregate complexes with RNA. Electron microscopic analysis showed that the aggregate complexes were comprised of filaments. These results indicate that not only modules but also secondary structure units dissected from a globular protein have functional and structure-forming capabilities.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ribonucleases/química , Ribonucleases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/ultraestrutura , Dobramento de Proteína , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , RNA Ribossômico 5S/metabolismo , RNA Ribossômico 5S/farmacologia , RNA Ribossômico 5S/ultraestrutura , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/genética , Ribonucleases/ultraestrutura , Deleção de Sequência/genética , Soluções , Relação Estrutura-Atividade , Temperatura , TrifluoretanolRESUMO
Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P. Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined. Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives. However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results. Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme. Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor. Further studies showed that the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure. The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer. Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition.
Assuntos
Endorribonucleases/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Catalítico/metabolismo , RNA de Transferência de Tirosina/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Núcleo Celular/enzimologia , Endorribonucleases/antagonistas & inibidores , Modelos Químicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Precursores de RNA/química , RNA Catalítico/antagonistas & inibidores , RNA de Cadeia Dupla/metabolismo , RNA Ribossômico 5S/farmacologia , RNA de Transferência/farmacologia , Ribonuclease P , Saccharomyces cerevisiae/enzimologia , Especificidade por SubstratoRESUMO
Mouse myeloma cells (SP2/0) were incubated with 125I-5s rRNA from rabbit reticulocytes and processed for autoradiography. The results indicated that 5s rRNA could pass into the nuclei of mouse myeloma cells. In a separate experiment, SP2/0 were incubated with cold 5s rRNA, then with 3H-TdR and processed for autoradiography. It was found that in the mouse myeloma cells, DNA synthesis and cell division were obviously suppressed. In another series of experiments, rRNA was extracted from rabbit bone marrow, reticulocytes and erythroid cells and from rat embryonic liver and erythroid cells. The rRNA was analyzed by agarose electrophoresis. It was found that the amount of 5s rRNA in various stages of erythroid development changed along with the denucleating process. Thus it seems likely that 5s rRNA from mammalian erythroid cells could play a role in reversing the malignant phenotype of tumor cells and denucleation of mammalian erythroid cells through inhibiting DNA synthesis.
